A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S.

Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling.

In this study, S. cerevisiae crude membrane fractions were prepared using the acid-labile detergent RapiGest from cells grown under rich and minimal media conditions using 14N and 15N ammonium sulfate as the sole nitrogen source. Four independent MudPIT analyses of 1:1 mixtures of sample were prepared and analyzed via quantitative multidimensional protein identification technology on a two-dimensional ion trap mass spectrometer.

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression.

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing.

Proteomic comparison of two fractions derived from the transsynaptic scaffold.

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.

Maspin alters the carcinoma proteome.

Maspin, a member of the serine protease inhibitor (serpin) family, is a tumor suppressor in breast and prostate cancer. To address molecular mechanisms underlying maspin's activity, we restored its expression in invasive carcinoma cells and analyzed the resulting changes by shotgun proteomics. Using a mass spectrometry-based multidimensional proteomic method, we observed changes to the expression of approximately 27% of the detectable proteome.

Identification of novel integral membrane proteins of the nuclear envelope with potential disease links using subtractive proteomics.

Lamin A and some integral membrane proteins of the nuclear envelope (NE) have been linked to human diseases, mostly dystrophies. To comprehensively identify integral membrane proteins specific to the nuclear envelope, we have carried out a subtractive proteomics analysis of NEs isolated from rodent liver using Multidimensional Protein Identification Technology (MudPIT). An NE fraction and a nucleus-depleted membrane fraction were separately analyzed by MudPIT and proteins appearing in both fractions were 'subtracted' from the NE fraction.

The mammalian Mediator complex.

The multiprotein Mediator (Med) complex is an evolutionarily conserved transcriptional regulator that plays important roles in activation and repression of RNA polymerase II transcription. Prior studies identified a set of more than twenty distinct polypeptides that compose the Saccharomyces cerevisiae Mediator. Here we discuss efforts to characterize the subunit composition and associated activities of the mammalian Med complex.

Characterization of the yeast trimeric-SAS acetyltransferase complex.

The yeast SAS2 (Something About Silencing 2) gene encodes a member of the MYST protein family of histone acetyltransferases (HATs) and is involved in transcriptional silencing at all silent loci (HML, HMR, telomeres, and rDNA) in Saccharomyces cerevisiae. Sas2 is the catalytic subunit of a yeast histone acetyltransferase complex termed SAS complex. The enzymatic activity of SAS complex on free histones has been reported, but nucleosomal HAT activity has not yet been documented.

The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex.

Covalent modifications of the histone tails and the cross talk between these modifications are hallmark features of gene regulation. The SAGA histone acetyltransferase complex is one of the most well-characterized complexes involved in these covalent modifications. The recent finding that the removal of the ubiquitin group from H2B is performed by a component of SAGA, Ubp8, is intriguing as it assigns two posttranslation modification processes to one complex.

The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes.

The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis. We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J.

A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.

Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific.

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei.

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp.

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions.

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair.

Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle.

To investigate the role of post-transcriptional controls in the regulation of protein expression for the malaria parasite, Plasmodium falciparum, we have compared mRNA transcript and protein abundance levels for seven different stages of the parasite life cycle. A moderately high positive relationship between mRNA and protein abundance was observed for these stages; the most common discrepancy was a delay between mRNA and protein accumulation. Potentially post-transcriptionally regulated genes are identified, and families of functionally related genes were observed to share similar patterns of mRNA and protein accumulation.

Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex.

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified.

Actin-binding proteins in a postsynaptic preparation: Lasp-1 is a component of central nervous system synapses and dendritic spines.

CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules.

Proteomics approach reveals novel proteins on the surface of malaria-infected erythrocytes.

Proteins on the surface of parasite-infected erythrocytes (PIESPs) have been one of the major focuses of malaria research due to their role in pathogenesis and their potential as targets for immunity and drug intervention. Despite intense scrutiny, only a few surface proteins have been identified and characterized. We report the identification of two novel surface proteins from Plasmodium falciparum-infected erythrocytes.

A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology.

The Mediator is a multiprotein transcriptional coactivator that is expressed ubiquitously in eukaryotes from yeast to mammals and is required for induction of RNA polymerase II (pol II) transcription by DNA binding transcription factors. In the work described here, we exploit multidimensional protein identification technology (MudPIT) to carry out a proteomic analysis of the subunit composition of the mammalian Mediator complex. By comparing MudPIT data sets obtained from six independent Mediator preparations immunoaffinity purified through their Nut2 (MED10), Med25 (MED9), Intersex (MED29), LCMR1 (MED19), AK007855 (MED28), or CRSP70 (MED26) subunits, we identify a set of consensus mammalian Mediator subunits.

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time.

Proteomics in malaria.

The recent completion of human, Anopheles gambiae, and Plasmodium falciparum genomes relevant to the study of human malaria allows the application of modern proteomic technologies to complement previously implemented conventional approaches. Proteomic analysis has been employed to elucidate global protein expression profiles, subcellular localization of gene products, and host-pathogen interactions that are central to disease pathogenesis and treatment. The high-throughput nature of these techniques is in accord with the pace of drug and vaccine development that have the potential to directly reduce the morbidity and mortality of disease.

The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion.

The first gene characterizing the clag (cytoadherence linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (Clag9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. Other genes related to clag9 have been identified on different chromosomes.

A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2.

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S.

Nuclear membrane proteins with potential disease links found by subtractive proteomics.

To comprehensively identify integral membrane proteins of the nuclear envelope (NE), we prepared separately NEs and organelles known to cofractionate with them from liver. Proteins detected by multidimensional protein identification technology in the cofractionating organelles were subtracted from the NE data set. In addition to all 13 known NE integral proteins, 67 uncharacterized open reading frames with predicted membrane-spanning regions were identified.

Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data.

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context of Plasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuated P.